Phosphors typically comprise one or more rare earth metals in a host material. Up-converter phosphors emit light in the visible wavelength radiation range (550-800 nanometers) when excited by long wavelength radiation, e.g., light in the infrared wavelength spectrum. This is accomplished by multiple absorption of infrared photons and energy transfer between the absorbing and emitting ions. For example, it is known that yttrium oxysulfide, Y.sub.2 O.sub.2 S, gadolinium oxysulfide, Gd.sub.2 O.sub.2 S, and lanthanum oxysulfide La.sub.2 O.sub.2 S, doped with certain activator couples, will be excited by 0.96 micron wavelength radiation. Such radiation can be provided by semiconductor lasers.
These phosphors have been tried as phosphorescent labels for biological assays. Detection methods for macromolecules such as proteins, drugs, polynucleotides and the like include an analytical reagent that binds to a specific target macromolecular species and produces a detectable signal, provided by a label, such as a radioisotope or covalently-linked fluorescent dye or phosphor. This process is shown in graphic form in FIG. 1, which shows attachment of a specific antigen to a microtiter plate surface, capture of an antibody by the antigen, and attachment of a phosphor label by the antigen/antibody. This attachment and the relative sizes of known phosphors and antibodies are further illustrated in FIG. 2.
Up-converting phosphors have several advantages over other known materials, such as radioisotopes and covalently-linked fluorescent dyes, for such label applications. Radioimmunoassays, while they are sensitive, use radioactive materials which are potential health hazards for the operators of the tests, and which in turn also require special handling and expensive disposal problems. Radioisotopes are unstable and they do not produce strong signals in the ultraviolet, infrared or visible portions of the electromagnetic spectrum, and thus cannot be used for methods including microscopy, image spectroscopy and flow cytometry that employ optical methods for detection of the label.
Fluorescent labels thus have come into widespread use for such methods, including small organic dye molecules which can be illuminated with light of a particular excitation frequency so that they give off emissions that can be detected by electro-optical sensors. However, these fluorescent dye labels have limited sensitivity because the specific fluorescent signal of the label is difficult to detect from nonspecific background fluorescent signals given off by other reagents required for the test, such as serum, fixatives and the sample itself, as well as autofluorescence in the visible wavelength range of optical lenses and excitation light reflected from the equipment used to carry out the test. As an example, whole blood samples strongly scatter light at short wavelengths of about 600 nm, which is also the emission range of fluorescent dye reporters. Thus such fluorescent dyes are not well suited for immunoassays of whole blood. Since fluorescent dyes have a short lifetime, about 1-100 nanoseconds, it is often difficult to measure the label light. Another disadvantage in the use of fluorescent dyes is that these dyes bleach out due to photolytic decomposition of the dye molecules during exposure to light.
The use of up-converting phosphors for immunoassays has been disclosed. Such phosphors can be excited by photons of a frequency which can be provided by inexpensive near-infrared laser diodes or light-emitting diodes for example, and they emit light of a lower frequency band, in the visible range. Thus the photons of the emitted radiation are of higher energy than the excitation energy, and the emitted radiation is "up-shifted" from the excitation radiation. Since background fluorescence in the visible range is negligible if near infrared excitation wavelength light is used, the use of up-converter labels provides an essentially background-free visible emission signal. The ability to use efficient laser diodes or light-emitting diodes (LEDs) reduces the system size, the power requirements and the costs required to perform the assay. Solid state diode lasers can be tailored to operate at any desired wavelength in the near infrared range, and since they are inexpensive they are compatible with a low cost assay kit.
The phosphors of the invention can be used as immunoassay labels by attaching them to one or more probes, such as antibodies, protein A, polypeptide ligands of cellular receptors, polynucleotides, drugs, antigens, toxins and the like. When they serve as a reporter, or a light detectable marker, attachment of the label can be accomplished in various known ways, for example by coating phosphor particles with a polycarboxylic acid whereby various probes will be physically adsorbed to the surface of the phosphor particles. Other attaching agents such as siloxanes are also well known.
Such phosphor particles are typically smaller than about 3 microns in diameter, but it is preferred that the phosphor particles be as small as possible while still generating a detectable signal. For particular tests, such as detection of an abundant nuclear antigen in a permeabilized cell, a small phosphor particle is required that can readily diffuse and penetrate subcellular structures. Further, since during the course of the assay bound and unbound phosphor particles must be separated and differentiated, it would be highly desirable that the phosphor particles be uniform in size. The size of the particles, their weight and their morphology are all important criteria because they affect the strength of particle binding and the specificity of the separation process. Further, since in an assay application each of the particles should have a like number of active binding sites, it is also desirable that the particles be of similar size.
Thus the as-formed phosphors are generally milled to reduce their particle size. Prior art milling methods employ milling in a conventional barrel mill with zirconia and/or alumina balls for up to 48 hours or longer. This produces phosphor particles of from 0.01 to 3 microns in size. If a particular particle size is desired, fractions of the desired particle size can be prepared by sedimentation which generally takes a day or more, and removal of the undesired (larger) particle sizes.
It is apparent that the milling and sedimentation processes are quite lengthy, which adds to the cost of preparing suitable phosphors for immunoassay labels. But more importantly, the milling process acts to fracture the large crystalline phosphor particles, forming irregularly shaped particles.
Thus, the milling process produces non-spherical particles and a quite large range of particle sizes, even after sedimentation. Thus it would be highly desirable to be able to produce phosphor particles having uniform, small particle size and spherical morphology.
Submicron particles of yttrium oxide have been made, doped with ytterbium and erbium [(Y.sub.0.86 Yb.sub.0.08 Er.sub.0.06).sub.2 O.sub.3 ]. This is a relatively efficient red up-converter phosphor, but it is slightly water sensitive, especially as small particles. Thus, in order to produce a more efficient luminescent material, it is annealed in air at 1500.degree. C. However, this annealing process forms aggregates of the phosphor, in which some of the particles grow together, and which are very difficult to break apart, even by sonication techniques. Further, when the oxide is converted to the corresponding oxysulfide for use as a phosphor label, the phosphor particles aggregate further and produce non-spherical particles of 1-3 microns or more in size.
Thus it would be highly desirable to be able to form yttrium or gadolinium oxysulfide phosphors having small uniform particle size wherein the particles are spherical.